This invention relates to methods and compounds for treating Alzheimer""s disease.
Most current thinking about Alzheimer""s disease suggests that the dementia characteristic of the disease results from aggregates formed in the brains of afflicted individuals. The aggregates are thought to be formed by a peptide of 40-43 amino acids called Axcex2, which is derived from the amyloid precursor protein.
The Axcex2 peptide is normally found in trace amounts in blood and in cerebrospinal fluid. However, it can be found in increased levels in these fluids in patients suffering from some forms of Alzheimer""s disease, and it is thought that increased concentrations of the Axcex2 peptide in these fluids contribute to its tendency to form aggregates. Because Alzheimer""s disease can take many years to become clinically manifest, it is believed that over time even very small increases in circulating or cerebral spinal fluid Axcex2 levels may be damaging.
We have discovered that the addition of Wnt1 protein to Axcex2 peptide-producing cells affects the levels of Axcex2 peptide produced by these cells. Accordingly, the invention features methods of screening for compounds able to bind to the Wnt receptor and decrease the amount of Axcex2 peptide produced by these cells. The invention also features methods for treating Alzheimer""s disease using compounds able to bind to the Wnt receptor and lower the amount of Axcex2 peptide produced.
In one aspect, the invention features a method of identifying a compound that inhibits expression of an Axcex2 peptide by contacting the compound with a Wnt receptor or a Wnt-binding fragment of Wnt receptor and determining the ability of the compound to bind to the Wnt receptor or Wnt-binding fragment of Wnt receptor. The binding of the compound is an indication that the compound lowers the level of Axcex2 peptide.
In one embodiment, the compound is a mammalian Wnt protein or a Wnt-receptor binding fragment of a mammalian Wnt protein, e.g., a human Wnt1 protein or a Wnt1-receptor binding fragment thereof. In another embodiment, the compound is a peptide mimetic of a Wnt protein. In yet another embodiment, the compound is a non-peptide mimetic of a Wnt protein.
In another aspect, the invention features a method of identifying a compound that inhibits expression of Axcex2 peptide by contacting a cell producing detectable levels of Axcex2 peptide with a Wnt antagonist and measuring the amount of Axcex2 peptide produced. A decrease in the amount of Axcex2 peptide produced by cell in the presence of the Wnt antagonist indicates that the Wnt antagonist is a compound that decreases Axcex2 peptide expression. The Wnt antagonist can include, e.g., a cysteine-rich domain (CRD)-containing fragment of the Wnt-receptor or the Fritz protein.
In another aspect, the invention features a method of identifying a compound that inhibits expression of Axcex2 peptide by (a) culturing a cell expressing a Wnt-receptor and Axcex2 peptide in the presence of the compound; and (b) evaluating the expression of Axcex2 peptide by the cell. A lowered level of Axcex2 peptide relative to a control cell indicates the compound inhibits expression of Axcex2 peptide.
In yet another aspect, the invention features a method of evaluating a compound for the ability to lower Axcex2 peptide levels in a mammal by contacting the compound with Wnt or a Wnt receptor-binding fragment of Wnt and Wnt-receptor or a Wnt binding fragment of Wnt receptor and determining the ability of the compound to enhance the binding of the Wnt or a Wnt receptor-binding fragment of Wnt with the Wnt receptor or a Wnt-binding fragment of Wnt receptor. An increase in the binding in the presence of the compound compared to the binding in the absence of the compound indicates that the compound lowers extracellular Axcex2 levels.
In a further aspect, the invention features a method for identifying candidate therapeutic agents for the treatment of Alzheimer""s disease by (a) providing a cell which expresses a mammalian Wnt receptor, the cell comprising a reporter construct, the reporter construct comprising a sequence encoding a detectable protein, the sequence encoding the detectable protein being operably linked to a Wnt receptor responsive regulatory element; (b) contacting the cell with a test compound; (c) measuring the expression of the detectable protein in the presence of the test compound. An increase in the expression of the detectable protein in the presence of the test compound compared to the absence of the test compound indicates that the test compound is a candidate therapeutic agent for treatment of Alzheimer disease.
In one embodiment, the detectable protein is xcex2-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase (neor, G418r), dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), lacZ (encoding xcex2-galactosidase), or xanthine guaninephosphoribosyltransferase (XGPRT). The reporter gene can be alkaline phosphatase.
In another aspect, the invention features a method of inhibiting Axcex2 expression in a cell by identifying a Wnt-responsive cell that expresses Axcex2 peptide and contacting the cell with a compound that binds to Wnt, or to a Wnt receptor. The binding of the compound to Wnt inhibits Axcex2 expression by the cell.
The cell can be in a mammal, e.g., in vivo, or can be a cultured cell, i.e., in vitro. The compound can inhibit secretion of Axcex2 peptide from the cell, or can inhibit intracellular accumulation of Axcex2 from the cell.
Preferably, the Wnt protein is a Wnt1 protein.
In another aspect the invention features a method of inhibiting Axcex2 expression in a cell by identifying a Wnt-responsive cell that expresses Axcex2 peptide, and contacting the cell with a compound that binds to a Wnt receptor, wherein binding of the compound to the Wnt receptor inhibits Axcex2 expression by the cell.
In a further aspect, the invention features a method of treating Alzheimer""s disease by introducing into a human suspected of needing treatment for Alzheimer""s disease an effective amount of a compound binding to the human Wnt receptor. Binding of the compound is associated with decreased levels of Axcex2 polypeptide levels in the patient.
In a still further aspect, the invention features a method of preventing Alzheimer""s disease by introducing into a human indicating as needing prophylactic treatment for Alzheimer""s disease an effective amount of a compound characterized by binding to the human Wnt receptor.
In a further aspect, the invention features a method of lowering Axcex2 peptide levels in an mammal by administering a compound characterized by binding to a Wnt receptor in an amount sufficient to lower Axcex2 peptide levels.
An xe2x80x9cAxcex2 peptidexe2x80x9d includes an extracellular fragment of the xcex2 amyloid precursor protein that is 40-43 amino acids in length and is detected extracellularly.
A xe2x80x9cWnt proteinxe2x80x9d includes a member of the Wnt family of proteins.
A xe2x80x9cWnt receptorxe2x80x9d includes a macromolecule, e.g., a protein, able to confer responsiveness to a Wnt ligand when expressed on the surface of a cell.
A xe2x80x9cpeptide mimeticxe2x80x9d includes a Wnt analog in which one or more peptide bonds have been replaced with an alternative type of covalent bond which is not susceptible to cleavage by peptidases.
A xe2x80x9cWntxe2x80x9d antagonist is a compound that inhibits the activity of a Wnt protein.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
The present invention is based on the discovery that the Wnt protein, when added to cells that secrete Axcex2 peptide, affects the amount of Axcex2 peptide secreted by these cells.
The Wnt protein is a secreted protein encoded by a family of conserved genes that have been identified as both proto-oncogenes and as having functions important in development (reviewed in Nusse et al., Cell 69:1073, (1992)).
The Wnt protein is thought to act through cellular receptors encoded by members of the frizzled gene family. A receptor protein able to confer responsiveness to Wg, the homolog of Wnt in Drosophila melanogaster, is encoded by the frizzled2 locus (xe2x80x9cDfz2xe2x80x9d) (Bhanot et al., Nature 382:224 (1996); see also Roush, Science 273:309 (1996)). The full-length Dfz2 cDNA (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) are shown in Table 1.
Dfz2 is a member of the frizzled (""fz) gene family, which is found in a wide variety of eukaryotes (Wang et al., J. Biol. Chem. 271:4468 (1996)). The Dfz2 gene has sequence features characteristic of this family: an amino terminal signal sequence precedes a domain characterized by ten invariant cysteine residues (the cysteine rich domain, or
xe2x80x9cCRDxe2x80x9d). The CRD is followed by a highly divergent region of 40-100 mainly hydrophilic residues encoding seven putative membrane-spanning segments (Bhanot et al.).
The present invention provides methods of screening for compounds that are able lower the levels of Axcex2 peptide by acting via the Wnt-receptor protein, and for methods of treating Alzheimer""s disease using these compounds. One way to identify compounds able to lower Axcex2 peptide levels is based on the interaction of the compound with the Wnt receptor, and/or compounds which activate other members of the Wnt signal transduction pathway.
Methods of Screening for Wnt-Receptor Binding Compounds Able To Lower Axcex2 Levels
Compounds able to lower levels of Axcex2 peptide can be identified based on their interaction with a Wnt receptor or a Wnt-binding fragment of a Wnt receptor.
Screening assays can be performed with a Wnt receptor, or a Wnt-binding fragment of a Wnt receptor. The Wnt receptor protein can be a member of the frizzled protein family, such as those described in Barnot et al., above. The screening assays can also be performed using the mammalian frizzled sequences Hfz5 (human), mouse Mfz4, Mfz7, or Mfz8 (mouse), or on cells expressing frizzled sequences. The nucleic acid sequences and predicted amino acid sequences of Hfz5, Mfz4, Mfz7, or Mfz8 are shown in Tables 2-5.
When the Wnt receptor is a frizzled protein, a Wnt-binding fragment of the receptor can include the CRD domain of a frizzled protein, e.g., the fragments summarized in FIG. 2 of Mayr et al., Mech. Dev. 63:109-125, 1997. These fragments include amino acids 1-264 (SEQ ID NO:15) of the human Hfz5 amino acid sequence shown in Table 2.
The Wnt receptor can alternatively be a member of the Notch family of proteins. Genetic and biochemical
studies have implicated products of the Notch locus as being involved in the reception of Wnt signals (Couso et al., Cell 79:259 (1994); Axelrod et al., Science 271:1826 (1996). When Notch family members are used, calcium may be added in the medium to facilitate binding (Ingham, Trends Genet. 12:382, 384 (1996)).
One way the Wnt receptor protein can be provided is on a cell responsive to Wnt. Several such cell lines are known, e.g., PC12 pheochromocytoma cells and C57MG mammary carcinoma cells.
Alternatively, the Wnt receptor can be introduced into a cell that is otherwise not responsive to Wnt using methods known in the art, e.g., as a polypeptide or by introducing cDNA encoding a Wnt receptor into an appropriate cell. Thus, the source of Wnt-responsive cell is not critical when compounds are screened using cell-based assays. For example, Barnot et al., supra, have demonstrated that the Dfz2 protein confers Wg-responsiveness on Drosophila S2 cells and human embryonic kidney cells.
Candidate compounds can be screened for binding to the Wnt receptor using methods known in the art. Binding may be assessed by examining compounds for the ability to stimulate the Wnt signal transduction pathway, e.g., by assessing stabilization of proteins in the xcex2-catenin family as is described in Bahnot et al. for the armadillo (Arm) protein. Another way in which Wnt activity can be assessed by measuring activation of the T cell factor lymphoid enhancer factor (TcF-Lef) (Behrens et al., Nature 382:638-42, 1996).
In some situations it may be desirable to first screen candidate compounds for direct binding to the Wnt receptor, or Wnt-binding fragments thereof, such as the CRD domain, in vitro, e.g., in a gel mobility shift assay and/or an immunoprecipitation assay, and then test Wnt-receptor binding proteins for the ability to lower Axcex2 levels in a subsequent step.
Levels of Axcex2 peptide can be measured using methods well-known in the art, e.g., the methods described in Haass et al., Nature 359:322-25, (1992).
The assays described herein are designed to identify compounds that interact with (e.g., bind to) a Wnt receptor. These compounds can include, but are not limited to, those that interact with members of the frizzled family, These compounds can also include those that interact with (e.g., bind to) intracellular proteins that interact with Wnt (including, but not limited to, the Wnt receptor) compounds that interfere with the interaction of Wnt with transmembrane or intracellular proteins involved in Wnt-mediated signal transduction, e.g., proteins analogous to the dishelved and Arm gene products, and to compounds that modulate the activity of a Wnt receptor gene (i.e., modulate the level of Wnt receptor gene expression). Assays can additionally be utilized which identify compounds that bind to Wnt receptor gene regulatory sequences (e.g., promoter sequences) and which may modulate Wnt receptor gene expression.
The compounds can also be those resembling, e.g., having homology to, the Wnt receptor, or fragments thereof, e.g., fragments containing the CRD-domain. Some examples of CRD domain-containing polypeptides are amino acids 1-348 (SEQ ID NO:16) of the Df2z homolog (SEQ ID NO:2) and amino acids 1-264 (SEQ ID NO:15) of the human Hfz5 homolog. An example of a compound of this type is the human Fritz protein (SEQ ID NO:14), which is a secreted protein having homology to the CRD-domain found in members of the frizzled family, and which has been reported to antagonize Wnt function (Lin et al., Proc. Natl. Acad. Sci. (USA) 94:11196-11200 (1997); Mayr et al., Mec. Dev. 63: 109-25 (1997)). Nucleic acids encoding human and mouse Fritz sequences, and the predicted amino acid sequences encoded by these sequences, are shown in Tables 6 and 7, respectively.
In general, compounds that can be screened in accordance with the invention include, but are not limited to polypeptides, peptides, antibodies and fragments thereof, and other organic compounds (e.g., peptidomimetics) that bind to the extra-cellular domain or cysteine-rich domain of a Wnt receptor and either mimic the activity triggered by the natural ligand (i.e., agonists) or inhibit the activity triggered by the natural ligand (i.e., antagonists); as well as peptides, antibodies or fragments thereof, and other organic compounds that binding to Wnt.
Such compounds can include, but are not limited to, peptides such as, for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g., Lam, et al., Nature 354:82-84, 1991; Houghten, et al., Nature 354:84-86, 1991), and combinatorial chemistry-derived molecular library made of D- and/or L-configuration amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell limited to, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(abxe2x80x2)2 and Fab expression library fragments, and epitope-binding fragments thereof), and small organic or inorganic molecules.
Other compounds that can be screened in accordance with the invention include but are not limited to small organic molecules that are able to cross the blood-brain
barrier, gain entry into an appropriate cell (e.g., a neuron, glial cell, microglial cell, or endothelial cell) and affect the expression of a gene involved in the Wnt signal transduction pathway (e.g., by interacting with the regulatory region or transcription factors involved in gene expression); or such compounds that affect the activity of Wnt protein to the Wnt receptor (e.g., by inhibiting or enhancing the enzymatic activity of the CD) or the activity of some other intracellular factor involved in the Wnt signal transduction pathway, such as, e.g., disheveled or delta-catenin.
Computer modelling and searching technologies permit identification of compounds, or the improvement of already identified compounds, that can modulate Wnt expression or activity. Having identified such a compound or composition, the active sites or regions are identified. Such active sites might typically be ligand binding sites, such as the interaction domains of Wnt with the Wnt receptor. The active site can be identified using methods known in the art including, for example, from the amino acid sequences of peptides, from the nucleotide sequences of nucleic acids, or from study of complexes of the relevant compound or composition with its natural ligand. In the latter case, chemical or X-ray crystallographic methods can be used to find the active site by finding where on the factor the complexed ligand is found.
Next, the three dimensional geometric structure of the active site is determined. This can be done by known methods, including X-ray crystallography, which can determine a complete molecular structure. On the other hand, solid or liquid phase NMR can be used to determine certain intra-molecular distances. Any other experimental method of structure determination can be used to obtain partial or complete geometric structures. The geometric structures can be measured with a complexed ligand, natural or artificial, which may increase the accuracy of the active site structure determined.
If an incomplete or insufficiently accurate structure is determined, the methods of computer based numerical modelling can be used to complete the structure or improve its accuracy. Any recognized modelling method may be used, including parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models. For most types of models, standard molecular force fields, representing the forces between constituent atoms and groups, are necessary, and can be selected from force fields known in physical chemistry. The incomplete or less accurate experimental structures can serve as constraints on the complete and more accurate structures computed by these modeling methods.
Finally, having determined the structure of the active site, either experimentally, by modeling, or by a combination, candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure. Such a search seeks compounds having structures that match the determined active site structure and that interact with the groups defining the active site. Such a search can be manual, but is preferably computer assisted. Compounds found from this search are potential Wnt modulating compounds.
Alternatively, these methods can be used to identify improved modulating compounds from an already known modulating compound or ligand. The composition of the known compound can be modified and the structural effects of modification can be determined using the experimental and computer modelling methods described above applied to the new composition. The altered structure is then compared to the active site structure of the compound to determine if an improved fit or interaction results. In this manner, systematic variations in composition, such as by varying side groups, can be quickly evaluated to obtain modified modulating compounds or ligands of improved specificity or activity.
Further experimental and computer modeling methods useful to identify modulating compounds based upon identification of the active sites of Wnt, the Wnt receptor, Wnt, and related transduction and transcription factors will be apparent to those of skill in the art.
Examples of molecular modelling systems are the CHARMm and QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMm performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modelling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other.
A number of articles review computer modelling of drugs interactive with specific proteins, such as Rotivinen, et al., 1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinaly and Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perry and Davies, OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989, Proc. R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a model receptor for nucleic acid components, Askew et al., 1989, J. Am. Chem. Soc. 111:1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to regions of DNA or RNA, once that region is identified.
Although described above with reference to design and generation of compounds which could alter binding, one could also screen libraries of known compounds, including natural products or synthetic chemicals, and biologically active materials, including proteins, for compounds which are inhibitors or activators.
Compounds identified through the assays described herein may be useful, for example, in lowering extracellular Axcex2 levels, and in treating Alzheimer""s disease.
Methods of Lowering Axcex2 Peptide Levels
The methods of the invention are useful in treating diseases characterized by high levels of Axcex2 peptide, e.g., Alzheimer""s disease. The invention provides methods of lowering Axcex2 levels by administering compounds, such as those discussed above, which interact with the Wnt protein and/or the Wnt receptor, or members of the Wnt signal transduction pathway to lower Axcex2 levels.
The term xe2x80x9cfragmentxe2x80x9d, as applied to a peptide, will ordinarily be at least about 10 amino acids, usually about 20 contiguous amino acids, preferably at least 40 contiguous amino acids, more preferably at least 50 contiguous amino acids, and most preferably at least about 60 to 80 or more contiguous amino acids in length. Such peptides can be generated by methods known to those skilled in the art, including proteolytic cleavage of the protein, de novo synthesis of the fragment, or genetic engineering.
Peptide analogs can differ from the native peptides of a Wnt protein by amino acid sequence, or by modifications which do not affect the sequence, or by both. Peptide analogs can have sequences which differ from the wild-type sequence (i.e., the sequence of the homologous portion of the naturally occurring peptide) only by conservative amino acid substitutions, preferably by only one, two, or three, substitutions, for example, substitution of one amino acid for another with similar characteristics (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the peptide""s biological activity.
Modifications (which do not normally alter primary sequence) include in vivo or in vitro chemical derivitization of peptides, e.g., acetylation or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a peptide during its synthesis and processing or in further processing steps, e.g., by exposing the peptide to enzymes which affect glycosylation e.g., mammalian glycosylating or deglycosylating enzymes. Also included are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
The invention includes analogs in which one or more peptide bonds have been replaced with an alternative type of covalent bond (a xe2x80x9cpeptide mimeticxe2x80x9d) which is not susceptible to cleavage by peptidases. Where proteolytic degradation of the peptides following injection into the subject is a problem, replacement of a particularly sensitive peptide bond with a noncleavable peptide mimetic will make the resulting peptide more stable and thus more useful as a therapeutic. Such mimetics, and methods of incorporating them into peptides, are well known in the art. Similarly, the replacement of an L-amino acid residue is a standard way of rendering the peptide less sensitive to proteolysis. Also useful are amino-terminal blocking groups such as t-butyloxycarbonyl, acetyl, theyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl, benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyazelayl, methoxyadipyl, methoxysuberyl, and 2,4,-dinitrophenyl. Blocking the charged amino- and carboxy-termini of the peptides would have the additional benefit of enhancing passage of the peptide through the hydrophobic cellular membrane and into the cell.
Modification of these peptides to improve penetration of the blood-brain barrier would also be useful. Peptides may be altered to increase lipophilicity (e.g. by esterification to a bulky lipophilic moiety such as cholesteryl) or to supply a cleavable xe2x80x9ctargetorxe2x80x9d moiety that enhances retention on the brain side of the barrier (Bodor et al., Science 1992, vol. 257, pp. 1698-1700). Alternatively, the peptide may be linked to an antibody specific for the transferrin receptor, in order to exploit that receptor""s role in transporting iron across the blood-brain barrier (Friden et al., Science, 1993, vol. 259, pp. 373-377).
Peptides may be administered to the patient intravenously in a pharmaceutically acceptable carrier such as physiological saline. Standard methods for intracellular delivery of peptides can be used, e.g. delivery via liposomes. Such methods are well known to those of ordinary skill in the art. The formulations of this invention are useful for parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.
Therapeutic administration of a peptide intracellularly can also be accomplished using gene therapy, wherein a nucleic acid which includes a promoter operatively linked to a sequence encoding a heterologous peptide is used to generate high-level expression of the peptide in cells transfected with the nucleic acid. DNA or isolated nucleic acid encoding peptides of the invention may be introduced into cells of the patient by standard vectors and/or gene delivery systems. Suitable gene delivery systems may include liposomes, receptor-mediated delivery systems, naked DNA, and viral vectors such as herpes viruses, retroviruses, and adenoviruses, among others.
Pharmaceutically acceptable carriers are biologically compatible vehicles which are suitable for administration to an animal: e.g., physiological saline. A therapeutically effective amount is an amount of the nucleic acid of the invention which is capable of producing a medically desirable result in a treated animal.
As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient""s size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Dosages for the compounds of the invention will vary, but a preferred dosage for intravenous administration is from approximately 106 to 1022 copies of the nucleic acid molecule in the case of gene therapy.